Porosity gradient electrophoresis gel

ABSTRACT

Describes a process for controlling the polymerization and cross-linked density of electrophoretic gel products useful for separation of bioorganic molecules, which process does not use initiators common to processes of the art. Electron beam polymerized gels afford the desired advantages of being ultra thin and having a high electrophoretic resolution with programmable porosity profiles.

FIELD OF THE INVENTION

This invention relates to the electrophoretic gel products of controlledcross-link density, useful for separation of bioorganic molecules, andto controlled, radiative processes for their production, which do notuse initiators common to processes of the art.

BACKGROUND OF THE INVENTION

Electrophoresis is based on the principle that charged molecules orsubstances will migrate when placed in an electric field. Since proteinsand other biopolymers (e.g., DNA, RNA, enzymes and carbohydrates) arecharged, they migrate at pH values other than their isoelectric point.The rate of migration depends, among other things, upon the chargedensity of the protein or biopolymer and the restrictive properties ofthe electrophoretic matrix. The higher the ratio of charge to mass thefaster the molecule will migrate.

In theory, separation of different proteins could be readily achieved infree solution provided that the molecules differed sufficiently in theircharge densities. However, in practice separations in free solution aredifficult to achieve. Heat produced during electrophoresis can causeconvection disturbances in the liquid medium distorting the proteinbands. Recognition of the individual proteins is compromised because thebands are constantly broadened by diffusion. This continues even afterelectrophoresis has been stopped. Therefore, electrophoresis in freesolution is rarely carried out. In practice various supporting media arecurrently used to minimize convection and diffusion, and to effectseparation both on the basis of size and of molecular charge.

Many support media for electrophoresis are in current use. The mostpopular are sheets of paper or cellulose acetate, silica gels, agarose,starch, and polyacrylamide. Paper, cellulose acetate, and thin-layersilica materials are relatively inert and serve mainly for support andto minimize convection. Separation of proteins using these materials isbased largely upon the charge density of the proteins at the pHselected.

On the other hand, starch, agarose and polyacrylamide gel materials notonly minimize convection and diffusion but also actively participate inthe separation process. These materials provide a porous medium in whichthe pore size can be controlled to approximate the size of the proteinmolecules being separated. In this way, molecular sieving occurs andprovides separation on the basis of both charge density and molecularsize.

The extent of molecular sieving is thought to depend on how closely thegel pore size approximates the size of the migrating particle. The poresize of agarose gels is sufficiently large that molecular sieving ofmost protein molecules is minimal and separation is based mainly oncharge density. In contrast, polyacrylamide gels can have pores thatmore closely approximate the size of protein molecules and so contributeto the molecular sieving effect. Polyacrylamide has the furtheradvantage of being a synthetic polymer which can be prepared in highlypurified form.

The ability to produce a wide range of gel pore sizes and to form poresize gradients within the gel are additional advantages ofpolyacrylamide. Control over pore size enables mixtures to be sieved onthe basis of molecular size and enables molecular weight determinationsto be performed. These determinations are especially accurate if theproteins are coated with a detergent such as sodium dodecyl sulfate(SDS) which neutralizes the effects of molecular charge. This techniqueis referred to as SDS-PAGE electrophoresis.

PORE GRADIENT GELS

Polyacrylamide gels can be made with a gradient of increasing acrylamideconcentration and hence decreasing pore size. These gels are now usedextensively instead of single concentration gels, both for analysis ofthe protein composition of samples and for molecular weight estimationusing SDS as a denaturing agent to render the proteins in a uniformcharge environment. Step gradients in which gels of differentconcentration are layered one upon the other have been used. They tendedto give artifactual multi-component bands at the interface betweenlayers. It is now common to use continuous acrylamide gradients. Theusual limits are 3-30% acrylamide in linear or non-linear gradients withthe particular range chosen depending upon the size of the proteins tobe fractionated. During electrophoresis in gradient gels, proteinsmigrate until the decreasing pore size impedes further progress. Oncethis "pore limit" is approached the protein banding pattern does notchange appreciably with time although migration does not ceasecompletely.

One of the main advantages of gradient gel electrophoresis is that themigrating proteins are continually entering areas of gel with decreasingpore size such that the advancing edge of the migrating protein zone isretarded more than the trailing edge, resulting in a marked sharpeningof the protein bands. In addition, the gradient in pore size increasesthe range of molecular weights which can be fractionated simultaneouslyon one gel. Therefore a gradient gel will not only fractionate a complexprotein mixture into sharper bands than is usually possible with a gelof uniform pore size, but also can permit the molecular weightestimation of almost all the components.

Native proteins can also be analyzed on gradient gels usingnon-dissociating buffers.

Pore gradient gels are conventionally prepared by mixing high and lowconcentration monomer solutions in order to produce a concentrationgradient of acrylamide in the gel molds. Using this type of approachboth linear and non-linear gradient shapes can be prepared with respectto pore size along the length of the gel.

In addition to the gradient in acrylamide concentration, a densitygradient of sucrose or glycerol is often included to minimize mixing byconvective disturbances caused by the heat of polymerization. Someworkers avoid the latter problem by including a gradient ofpolymerization catalyst to ensure that polymerization occurs first atthe top of the gel and then proceeds to the bottom.

The polyacrylamide gel so prepared results from polymerization ofacrylamide and simultaneous polymer cross-linking by bifunctionalcompounds such as N,N'-methylene-bis-acrylamide (BIS). Thepolymerization is normally initiated by either ammonium persulfate orriboflavin. Thermal polymerization with persulfate is accelerated by theaddition of organic bases such as N,N,N',N'-tetramethylethylenediamine(TMEDA). Photochemical initiated polymerization by riboflavin requiresvisible or UV light. In all cases, oxygen inhibits the radicalpolymerization and monomer mixtures must be degassed prior toinitiation.

The details of the preparation and the use of such gels forelectrophoresis are generally and comprehensively reviewed by B. D.Hames in B. D. Hames and D. Rickwood, Eds., "Gel Electrophoresis ofProteins", pp. 1-89, IRL Press, Washington, D.C. (1981).

pH Gradient Gels (IEF)

Amphoteric materials (low molecular weight ampholytes) can be added togel formulations. Following polymerization, the ampholyte materialsmigrate in the electric field according to their pI (isoelectric points)and come to rest in zones in the order of their pI. A pH gradient isthus produced in the gel.

The technique of isoelectric focusing makes use of these gels and takesadvantage of the fact that each protein has a different pH at which itis electrically neutral--its isoelectric point (pI). Proteins areseparated according to pI by electrophoresis on a gel in which a stablepH gradient has been generated, extending from a low pH at the anode toa high pH at the cathode. For example, if proteins are applied to thegel at a given pH location, those with a higher pI will bear a netpositive charge and those with a lower pI will bear a net negativecharge. When an electric field is applied, the positively chargedmolecules will move towards the cathode into a zone of increasing pHwhile the negatively charged molecules will move towards the anode intoa zone of decreasing pH. When each protein reaches neutrality at its pI,it loses its electrophoretic mobility and becomes "focused" in a narrowzone.

Since diffusion is offset by the electric field, the bands do notbroaden as in other separation methods. Isoelectric focusing can resolveproteins that differ in pI by as little as 0.01 pH units.

Further details of IEF separations are described by B. An der Lan and A.Chrambach, in B. D. Hames and D. Rickwood, "Gel Electrophoresis ofProteins", pp. 157-186, IRL Press, Washington, D.C. (1981).

Note that much of the general literature describe gels of 500μ to 1500μthickness. However, selected works do disclose thin and ultrathin gelsin the range of 20μ to 500μ thicknesses. B. J. Radola and referencestherein, in: B. J. Radola, Ed., "Electrophoresis '79", Walter DeGruyter, New York (1980), pp 79-94 discuss ultrathin-layer isoelectricfocusing in 50-100μ polyacrylamide gels (no gradient) prepared bycasting on silanized glass or polyester sheets. B. J. Radola, A.Kinzkofer, and M. Frey in: R. C. Allen and P. Arnaud, Eds.,"Electrophoresis '81", Walter De Gruyter, New York (1981), pp 181-189describe isoelectric focusing in ultrathin-layer (20-50μ) polyacrylamidegels. No discussion appears as to the gel preparation. A. Gorg, W.Postel, R. Westermeier, E. Gianazza, and P. G. Righetti, ibid., pp.259-270 also describe isoelectric focusing and gradient electrophoresisin 240-360μ thick polyacrylamide gels. The gels are cast vertically, oneat a time, by gradient mixing of solutions to form the pore-sizegradient. Advantages of ultrathin gels are discussed.

Gels prepared by any of the above processes can suffer from severaldisadvantages which can compromise their utility in polyacrylamide gelelectrophoresis (PAGE) and in isoelectric focusing (IEF). Unreactedpolymerization initiators which are present can react with biologicalmolecules and cause distorted separations are sample decomposition. Theinitiators present can increase background staining and thus decreasecontrast of background with sample spots. Accelerators (e.g. TMEDA,tetramethylethylenediamine) can react with protein samples; canadversely affect electric conductivity of the gel matrix; and in IEF,may locally distort the pH gradients.

Prior to complete polymerization, the monomer solutions for both thickand thin gel preparation can be subject to distortion by thermalconvection, turbulent mixing and adverse capillary flow, all of whichcan distort or destroy the pore-size (concentration) gradient orgradient shapes. Such random errors reduce accuracy and reproducibilityof gradient gels. Thick gels require longer separation times and higherpower for separation which increases gel and sample heating. Botheffects can cause sample distortion or decomposition. The gels areusually prepared in small batches and are subject to the usualvariability of batch operations such as variable accuracy,reproducibility, and quality control.

The types of gels that can be prepared are limited. There is no"fine-tuning" control of these processes.

Numerous references further discuss modifications of the basicelectrophoretic and isoelectric focusing gels. U.S. Pat. No. 3,578,604discloses the preparation of non-gradient, acrylamide/agarose gels bycasting in the presence of persulfate. The use of photocatalysis isdisclosed by other art. The final gels were useful in electrophoreticseparations. Similarly, U.S. Pat. Nos. 3,788,950 and 4,189,370 claimprocesses for preparing acrylamide gels (or modifications thereof) inwhich gel formation is photoinitiated.

In general, the electrophilic gels prepared using the various prior artmethods suffer from many disadvantages. Among these, the presence ofvarious initiators in a gel often caused random reactions of theinitiator with free monomer, buffers or acrylamide polymer. Furthermore,the initiator or its by-products may react with the protein samplesthemselves, thereby distorting the electrophoretic results. Because ofthe ineffective mechanical blending of reagents and uncontrolledreactions, the gels produced by the techniques of the prior art areneither accurate nor are they highly reproducible. Another problemencountered with the prior art techniques is that because of the thermalconvection, vibration, mixing and capillary action, it is relativelydifficult to produce thin gels, i.e., those less than 500μ in thickness.Further, these prior art techniques tend to be relatively expensivesince they are batch-type operations and labor-intensive.

RADIATION POLYMERIZATION

In order to compensate for these disadvantages other types ofpolymerization processes have been reported in the literature. Theseinclude both thermal and photopolymerization processes.

The present invention makes use of radiation polymerization to improvein the control of the polymerization process.

The use of ionizing radiation (i.e. electron beams, α,β- and γ-rays,protons, x-rays) to polymerize acrylamide is known for solution andsolid systems. Chapiro ("Radiation Chemistry of Polymer Systems",Interscience, N.Y. (1962) pp 323-328) reviewed the general literature upto 1961.

R. Azzam and K. Singer, Prepr. Short Contrib.-Bratislava-IUPAC Int.Conf. Modif. Polym., 5th, 1, 143-148 (1979) discuss the effects of doserate, added species, and gel formation on the yield and MW ofpolyacrylamide prepared by radiation-induced polymerization. Thereaction was carried out in deoxygenated water with dose rates ofapproximately 5 Mrads/sec. The added species were primarily inorganicsalts. G. P. Korneeva, D. M. Margolin, E. B. Mamin and L. V. Chepel,Radiats. Khim., 2, 275-277 (1973) describe the use of 2.1 MeV electronsto prepare thin, pseudo-solid layers of polyacrylamide.

U.S. Pat. No. 3,993,551 discloses the radiation crosslinking ofpolyethylene oxide with at least another water soluble polymer in anaqueous system. The products are insoluble, hydrophilic gels whichcontain aqueous fluids and are useful as absorbing media. The preferredreaction uses 0.1 MeV to 20 MeV energy levels of radiation with totaldoses of 0.05 to 10 Mrads. One of the co-crosslinked, water solublepolymers is polyacrylamide. A variation in dose was noted to directlychange polymer absorbency.

U.S. Pat. No. 4,113,912 discloses hydrophilic porous structurescomprising a fluorocarbon resin structure containing awater-insolubilized polymer; in one case the latter polymer ispolyacrylamide crosslinked to form a swollen gel. One process, claimedfor producing the structures, utilizes ionizing radiation for theinsolubilization. Electron beams at 1-12 Mrads dosage were used as theionizing radiation. E. Collinson, F. S. Dainton, and G. S. McNaughton,Faraday Soc. Trans., 53, 476-488 (1957) describe in detail the x-ray andγ-ray initiated polymerization of acrylamide in aqueous solution. Therelationships of degree of polymerization (average) and of overallpolymerization rate to initial monomer concentration and dose rate aredescribed. The overall polymerization rate was found to be proportionalto the square root of the dose rate. Oxygen was reported to inhibit theacrylamide polymerization. Further, polymerization was reported tocontinue following termination of irradiation.

A Chapiro, "Radiation Chemistry of Polymeric Substances", High PolymerSeries, Vol. XV, Interscience, N.Y. 1962, pp 8-9 describes work done byGeneral Electric Co. and High Voltage Engineering, Inc. utilizing aresonant transformer and an experimental conveyor belt system for sampleirradiation. The dosage received by the sample depends on the beamcurrent, the scan width (and rate), and the speed of the conveyor belt.J. D. Nordstrom, in R. Bakish, Ed., "Electron and Ion Beam Science andTechnology--4th Intl. Conf.", The Electrochemical Soc., N.Y., 1970, pp605-618, reports that A. S. Hoffman and D. E. Smith, ACS PhiladelphiaLocal Section, November 1965 studied the conversion of monomer topolymer and found that gel formation (by cross-linking) continues toincrease with dose after all of the monomer is incorporated into thepolymer.

None of the polymers so produced were useful as electrophoretic gelproducts and none were prepared in a controlled, precisely programmedfashion to provide a controlled porosity.

The prior art provides important insight into the chemical events whichoccur in radiation induced polymerizations. With respect to theinvention described herein, the art is specifically helpful in tworespects.

First, the similarities and differences between chemical and radiationinduced polymerizations have been identified. For example, many of thereactions which occur in radiation induced polymerization are similar tothose which take place with chemical initiators. However, there areimportant differences which are unique to radiation polymerizationprocesses and can significantly alter the properties of the resultingpolymers. For example, radiation induced chain-to-chain cross-linking,chain to monomer cross-links, and chain degradation are importantcompeting reactions which normally cannot be achieved with conventionalchemical induced polymerization processes. The physical properties ofthe resulting polymers are thus not identical and, in many instances,can be substantially different.

Secondly, the prior art provides examples of the reactivity of theacrylamide monomer to radiation. Much of that work has only been carriedout for homopolymers where acrylamide is the only monomer. There iscurrently no precedent for this type of polymer being useful forelectrophoretic resolution of biopolymers. Furthermore, therelationships established between radiation exposure and the variousphysical properties of the polymers are not applicable as a guide sincethere currently are no known theoretical or emperical relationshipsestablished between the parameters investigated in those papers and theelectrophoretic properties of the resulting polymer solutions.

SUMMARY OF THE INVENTION

According to the present invention, a porous gel product useful forelectrophoretic separations is obtained. This product is characterizedby absence of an initiator or electron donor and is stable, reproducibleand has a controlled electrophoretic porosity. The gel product consistsessentially of polyacrylamide and water, but may also containacrylamide, agarose, bisacrylamide, and other monomers or polymers.Buffers or ampholytes, detergents and solutes may also be included inthe formulations. The formulations are polymerized and cross-linked in adefined manner which may be mathematically defined for a specific use.Gel products may vary in thickness from 50μ to 2 ml with the 100-300μrange being most preferred.

Preferably the gel product consists of an aqueous-swelled porous matrixformed from polymerized and cross-linked acrylamide monomers. Theconcentration of acrylamide monomers in the solutions from which gelsare made is essentially from about 3% (weight/volume) to about 30%(weight/volume). The gel products may have length, width and thicknessdimensions and the pore size of the gels may be in the form of agradient, which may vary along any of these dimensions and may be in alinear, or a complex function of pore size.

According to the method of this invention, charged bioorganic moleculesare electrophoretically separated using the gel product set forth inClaim 1, by the steps of placing a sample of bioorganic molecules on athin plate of the gel product and applying a voltage across a dimensionof the product.

The gel product is prepared by forming a water solution of a mixture offrom about 3% (weight/volume) to about 30% (weight/volume) of acrylamidemonomer and a cross-linking agent comprising about 0% (weight/weight) toabout 10% (weight/weight) of the total monomer, adding an aqueous bufferto the solution to adjust the pH and ionic strength, forming a solutioninto the shape of the desired gel product, and subjecting the solutionto ionizing radiation to polymerize and cross-link the monomer solution.The cross-linking agent includesN,N'-(1,2-dihydroxyethylene)-bis-acrylamide,N,N'-methylene-bis-acrylamide (BIS), N,N'-diallyltartramide (DATD),ethylenediacrylate, and N,N'-bis-acrylylcystamine and othercross-linking monomers. For a given composition, the dose and dose rateof the ionizing radiation applied to the monomer solution is regulatedto vary the gel porosity. The dose regulations is accomplished bymodulating the radiation flux as a function of the length, width andthickness of the gel. The flux may be modulated for fixed sheet-likegels by a computer and microprocessor programmed, moving shutter or byelectronically modulating or scanning the beam position. Alternatively,the radiation flux can be modified for continuous manufacture byconveying or moving individual gel molds or continuous or undivided gelcompositions under screens, halftone filters, grids, or variable shapedapertures, which modulate the radiation dose. Alternatively, electronicmodulation of the beam may also be used in the continuous process.

The gel products prepared in the manner described have many advantages,among these are enhanced electrophoretic properties. For example, thegradient can be versitally programmed and can be accurately and reliablyreproduced. The gels are cleaner due to the absence of initiator.Furthermore, thinner gels are readily produced using the method of thisinvention as well as custom gels, i.e., gells having any patternporosity profile desired. In general, cheaper gels may be producedbecause of the continuous process permitted by the method of thisinvention. The gels are safer and the user does not have to handle toxicacrylamide, only the packaged gel. Furthermore, the gels manufactured bythis invention can enable the application of higher voltages with fastermigration times. Additionally, the gel products of this invention havereduced endosmosis flow due to the reduced ionic content of the gelformulation.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed operation of the method described briefly above can be bestunderstood by reference to the following drawings in which:

FIG. 1 is a block diagram of the method for generating electrophoreticgels via ionizing radiation;

FIG. 2 is a drawing of an electron beam exposure pattern produced at 60cm below the accelerator tube window of a 2 MEV-G.E. ResonantTransformer;

FIG. 3 is a drawing illustrating the relationship between Log Rf andPercent Acrylamide for various proteins with respect to tracking dyes;

FIG. 4 is a drawing illustrating the effective porosity (T) versus Dose(D) as a function of Dose Rate (DR) for a given gel composition;

FIG. 5 is a drawing representing the rate of change of porosity (T) withrespect to time and dose as a function of dose and dose rate;

FIG. 6 is a drawing illustrating the limiting Dose (D) versus proteinMolecular Weight exclusion limit (MW_(o)) for a given dose rate and gelcomposition;

FIG. 7 is a top plan view of a stepped dose gradient slit;

FIG. 8 is a drawing illustrating the resulting gel porosity profile fromthe stepped dose gradient slit in FIG. 7;

FIGS. 9a-d is a drawing illustrating different typical slits used forporosity gradients;

FIG. 10 is a drawing illustrating a halftone electron filter for a loggradient porosity profile;

FIG. 11 is a drawing illustrating the electron scattering effect createdby the halftone electronic filter illustrated in FIG. 10;

FIG. 12 is a top plan view of a gel mold and film support;

FIG. 13 is a cross-sectional view in elevation taken along line 13--13of FIG. 12;

FIG. 14 is a top plan view of a gel mold and film support assembled andmounted on an aluminum support;

FIG. 15 is a cross-sectional view in elevation taken along line 15--15of FIG. 14;

FIG. 16 is a drawing of a shutter system for use in the manufacturing ofelectrophoretic gels;

FIG. 17 is a drawing illustrating the top view of a shutter system foruse in the manufacturing of electrophoretic gels;

FIG. 18 is a drawing of a conveyor system for use in the manufacturingof electrophoretic gels;

FIG. 19 is a drawing illustrating a device which is used to coolelectrophoretic gels during irradiation;

FIG. 20 is a drawing illustrating a typical slit, assembly used duringgel irradiation;

FIG. 21 is a drawing illustrating the top view of the slit shown in FIG.20;

FIG. 22 is a drawing illustrating a slit profile which yields a linearMW versus distance porosity profile;

FIG. 23 is a drawing illustrating the migration distance versus thelinear molecular weight which results from the dose profile created bythe slit in FIG. 22;

FIG. 24 is a drawing illustrating a slit profile which yields a log (MW)versus distance porosity gradient;

FIG. 25 is a drawing illustrating the migration distance versus log (MW)which results from the dose profile created by the slit in FIG. 24;

FIG. 26 is a drawing illustrating the percent residual monomer and gelporosity as a function of electron dose for a 20T, 1C Gel at 0.01Mrad/sec;

FIG. 27 is a drawing illustrating the effective percent T versus theadsorbed Dose for a 20T, 0C, 20T, 0.5C, and 20T, 1C Gels;

FIG. 28 is a drawing illustrating a continuous coating procedureutilizing a cover sheet; and

FIG. 29 is a drawing illustrating a continuous coating andpolymerization procedure utilizing an inert coating-radiation chamber.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT METHOD OF THE INVENTION

The method of the invention which overcomes many of the difficultiesexperienced in the prior art, is the ability to produce anelectrophoretic gel of a known porosity profile without the aid ofchemical initiators. This invention utilizes ionizing radiation as ameans for the initiation of radicals in the gel which in turn results ingel polymerization. The degree of polymerization and cross-linking of agiven monomer formulation in turn defines the electrophoretic gelseparation characteristics which are the key to protein separation inelectrophoretic gels. This radiation polymerization technology enablesthe preparation of all types of polyacrylamide electrophoresis gels.This technology enables control over the porosity and electrophoreticproperties of the resulting gel materials which to date has beenunobtainable. Using this approach, different types of electrophoresisgels can be successfully developed for a broad range of analyticalapplications including pore gradient gels for molecular weightdetermination and pH gradient gels for resolution on the basis ofmolecular charge and isoelectric point.

Referring to FIG. 1, there is seen the steps of the method of thisinvention which may be used to produce electrophoretic gel productscharacterized by an absence of initiators which consists essentially ofan aqueous-swelled porous matrix formed from polymerized andcross-linked monomers whereby the polymerization is initiated byradicals produced by ionizing radiation. This system illustrates themethod of the invention and details those essential elements which mustbe considered when engaging in the manufacture of electrophoretic gelproducts via electron beam radiation. In FIG. 1, steps 20 through 100specifically define these essential elements which in combinationprovides a unique approach in programming the porosity of a given gelcomposition. The steps are now described.

First the gel is formulated in step 20 by selecting the gel componentswhich, depending upon the chemical compositions chosen, influence theability of the gel materials to polymerize and cross-link to yield thedesired gel porosity and influence other separation characteristics. Inorder to establish a given radiation dose and dose rate combinationduring gel manufacturing the electron beam characteristics 30 must beknown. To help direct the radiation to the target in order to establishthe desired beam scanning patterns and dose profiles, beam modulation 40techniques have been developed which can in turn allow for complex gelporosity patterns to be established. Once the gel formulation 20,electrical beam characteristics 30, and beam modulation 40 have beenestablished, the next step involves the calibration of the selectedelectrophoretic gel compositions to the radiation system, which istermed calibration of electrophoretic response to radiation exposure 50.

A series of empirical calibration routines have been developed whichcharacterize the relationships of the radiation dose to gel porosity.These relationships can then be used to determine other relationshipswhich describes the effect of radiation dose to the molecular weight ofproteins as a function of distance across a given gel of knowncomposition. The calibration between radiation exposure and gelexclusion limit 60 can then be used for the generation of dose gradients80 which are linear or non-linear, logrithmic, or complex providing theability to program any combination of molecular weight or porosityversus distance relationship desired. This ability to control the gelporosity as a function of electron beam radiation produces a stable,accurate and reproducable gradient which can be easily adapted to acontinuous manufacturing operation. Therefore, production considerations90 are warranted. All of the above steps lead to the product 100 of thisinvention.

GEL FORMULATION 20

When considering manufacturing electrophoretic gels via ionizingradiation in accordance with this invention, the chemical compositionconsiderations (Step 20) are important in order to achieve the desiredporosity profile. For instance, the selection of gel materialsinfluences gel characteristics such as the specific or non-specificbinding of proteins or the electro endosmosis.

Acrylamide is the preferred and major monomer used in gel manufacturing;however, other water soluble monomers which can undergo radicalinitiated polymerization can be used. Other water soluble monomers maybe co-polymerized with acrylamide to achieve desired properties. By theprocess disclosed, certain water soluble polymers can also be used inconjunction with the above monomers or directly to form gel products.

The concentration of the above described monomers in the initial gelformulation and in the final gel products may vary from about 3% wt/v toabout 30% wt/v. This range is preferred for reasons of gel stability,strength and porous matrix properties. Gels with a low concentration ofacrylamide are the most porous and pass large molecules easily. Theseare useful in IEF where no restriction of molecules is desired or inseparation of large DNA fragments. Gels with a higher concentration ofacrylamide are less porous and provide restrictive passage for higherresolution of low molecular weight materials.

Chemical cross-linking can occur in the gels by the presence of suitablepolyunsaturated, functional acrylic or allylic compounds.

Compounds which act as suitable cross-linking agents include forexample, N,N'-methylene-bis-acrylamide (BIS), N,N'-diallyltartramide(DATD), ethylenediacrylate, N,N'-bis-acrylylcystamine,N,N'-(1,2-dihydroxyethylene)bisacrylamide and TEOTA(polyoxyethyltrimethylolpropanetriacylate). BIS is preferred for reasonsof reactivity, compatability and solubility but other cross-linkingagents may be added to or used instead of the BIS. They are added to theinitial gel formulation in concentrations of about 0% of the totalmonomer concentration (wt/wt); to about 10% of the total monomerconcentration (wt/wt); the actual amount being determined by the degreeof cross-linking required in the gel and may be determined empirically.The preferred range is about 2-7%. If these comonomers are added to theprimary acrylamide composition, the concentration of acrylamide may notbe less than about 50% of the total monomer concentration.

The total monomer concentration of the initial formulation (includingcross-linking agent) on a weight percent/volume basis is expressed as%T. The concentration of the cross-linking monomer is expressed as awt/wt percentage of the total monomer concentration and is called %C.

The gel formulations may be modified by the addition of polymericmaterials compatible with the electrophoretic separation technique. Suchcompounds include but are not limited to agarose, agar-agar, andpolyacrylamide (of varying molecular weights). The compounds may beadded to modify viscosity, porosity and gel strength. They can be addedto the formulation in concentrations ranging from about 0% wt/v to about20% wt/v, the preferred range is determined empirically on the basis ofeach individual gel product.

Water soluble polymers that may be useful individually or asco-components in gel formulation include polyacrylamide,polyvinylpyrrolidone, polyethylene oxide, polymethylvinylether,polyvinylalcohol and agarose.

The initial gel formulation and hence the final gel itself may befurther modified by the addition of varying concentrations of buffers,detergents, denaturants, ampholytes, and solutes. Buffer systems usedare dependent on the final end use of the gel product. Typical examplesof individual buffers, used in aqueous systems, includetris-(hydroxymethyl)aminomethane (TRIS)/hydrochloric acid, citric acid,sodium hydrogen phosphate, and borates. Buffers may be used individuallyor in combination to insure proper buffering capacity and ionicstrength. The concentrations and mixtures used in such buffer systemsare selected on the basis of final end-use and are obvious to oneskilled in the art. Details may be found in general references such asHames and Rickwood.

Detergents may be added to gel formulations or directly to thebio-organic sample to be separated. In both cases, the detergent isadded to solubilize the sample or to maintain a uniform charge to massratio so that samples separate solely on the basis of size. Detergentsand their concentrations are selected empirically on the basis of gelproduct end-use and on the basis of sample type to be separated.Detergents which may be used include, for example, cetyltrimethylammonium bromide, cetylpyridinium chloride, deoxycholate, sodium dodecylsulfate (SDS), polyethylene oxide sorbitan monooleate and ethoxylatedoctylphenols. General references such as Hames and Rickwood treat indetail the use of such detergents.

If the gel products are to be utilized for IEF separations, ampholytesmust be added to the gel formulation. Ampholytes are amphotericelectrolytes added to IEF gels to generate the pH gradient necessary forIEF separation. The ampholytes sold commercially are generally complexmixtures of polybasic amines and polyacids. There are no limitationsimposed by the instant invention on the type or concentration ofampholytes added to the instant gel products. References such as Hamesand Rickwood clearly define the limitations of ampholytes inherent inall gel mixtures.

The initial gel formulations of the instant invention may or may not bedegassed by inert gas (nitrogen, argon, helium, etc.) purging to removedissolved oxygen prior to casting. Purging is preferred. The use of highintensity ionizing radiation for polymerization and cross-linkingrapidly depletes dissolved oxygen and thus obviates the usual need torigorously exclude oxygen from the initial gel formulation as long assample preparation and exposure conditions remain constant andconsistent. However, during irradiation a cover sheet or innet exposurechamber is necessary to prevent inhibition of polymerization due toatmospheric oxygen, which can be rapidly replenished by diffusion.

Once mixed, the initial gel formulation may be modified with furtheradditives, if desired, and then is placed in a casting mold or simplybetween two cover sheets. Details of the casting are described laterunder production considerations 90.

Typical electron beam SDS-PAGE formulation for gradient and uniform gelsare set forth in Example 1, and for IEF gels in Example 2.

EXAMPLE 1

The following example illustrates the preparation of electrophoresisgels using electron beam radiation.

Two stock reagent solutions were prepared. In solution I, purifiedacrylamide (20 gm) and N,N'-methylene-bis-acrylamide (1.0 gm) weredissolved in purified water (75 ml). Solution II was prepared bydissolving tris(hydroxymethyl)aminomethane (18.1 gm), sodium dodecylsulfate (0.4 gm) in purified water (90 ml). The pH of the solution wasadjusted to pH 8.8 using HCl and then made up to a final volume of 100ml with purified H₂ O.

The gel formulation was then prepared by mixing 3 parts of theacrylamide stock solution (I) with 1 part of the TRIS buffer solution(II) yielding the following composition:

    ______________________________________                                        Acrylamide          20% weight percent                                        Bisacrylamide       5% by weight of the                                                           total monomer                                             TRIS Buffer         (.374 M)                                                  Sodium Dodecylsulfate                                                                             0.1%                                                      ______________________________________                                    

The gel formulation was then injected into molds and irradiated with 2MeV electrons resulting in absorbed radiation doses ranging from 0.03 to1.0 Mrads. The resulting gels on electrophoresis, exhibited porositiesequivalent to conventional control gels prepared with monomerconcentrations ranging from 5 to 28%T.

Exposures were performed at a distance of 60 cm using a dose rate of0.00856 Mrads/sec using the computer driven shutter system.

EXAMPLE 2

    ______________________________________                                        5.9T, 3C FORMULATION                                                          FOR ELECTRON BEAM IEF GELS                                                    ______________________________________                                        48% Acrylamide Stock Solution:                                                23.30 g recrystallized acrylamide                                             0.70 g BIS                                                                    dilute to 50 ml with H.sub.2 O                                                5,9T, 3C IEF Solution:                                                        11 ml 48% acrylamide stock solution                                           10 ml Servalyte 3-10 ampholytes*                                              69 ml H.sub.2 O                                                               ______________________________________                                         *or a mixture of standard ampholytes solution to equal 10 ml.            

ELECTRON BEAM CHARACTERISTICS 30 AND BEAM MODULATION 40

In order to successfully control the porosity across an electrophoreticgel product, certain electron beam characteristics must be considered ashigh-lighted below:

1. Beam Energy (Electron Volts e.v.)--Affects the penetration andvertical dose profile of ionizing radiation.

2. Incident Beam Power Flux (watts/cm²)--beam output power.

3. Rate of energy absorption (Mrads/sec) in the sample is related to themass thickness and beam energy and Power Flux obtained from dosimetrystudies.

4. Uniformity of The Resultant Electron Beam Pattern--This must be takeninto consideration in designing the slits, halftone filter or shutterprogram to compensate for non-uniformity.

The technique used to establish the above characteristics are well knownin the art. For example devices such as a Faraday Cup, can be used togather exposure distribution information as a function of a given X,Ycoordinate. From this information a radiation exposure map can begenerated as illustrated in FIG. 2. This exposure map, taken at adistance of 60 cm below the accelerator window, shows a uniform powerflux (watts/cm²) covering an area of 28 cm×25 cm in the center region ofthe beam. As will be described the electron beam is repetitively scannedin much the same manner as a TV raster scan, across the gel. Thisexposure map represents the output from a General Electric 2 MEVResonant Transformer.

Although an electron beam was used as the source of radiation in thisembodiment, other types of ionizing radiation such as positive ions,Alpha (α), Beta (β), Gamma (γ), and X-rays can be used.

Once the beam has been characterized, the dose of ionizing radiation canbe regulated to vary the gel porosity. The dose regulation isaccomplished by modulating the radiation flux (watts/cm²) as a functionof the length, width and thickness of the gel. The flux may be modifiedfor a fixed sheet-like gel by a programmed shutter or by electronicallymodulating the beam position, as in raster scanning of a televisionpicture. The instrument used to produce the ionizing radiation in thisembodiment utilized a pulse-beam raster scanning technique andtherefore, all calibrations were based upon this beam pattern. Where aconveyed web or moving sheet-like gel production operation is desired,such devices as halftone filters, grids, variable shaped apertures, orelectronic modulation of the flux beam position can be incorporated tomodify the flux. In a fixed gel production operation, a computer drivenshutter, halftone filters, grids, or electronic modulation of the fluxbeam position can also be incorporated. More detailed discussion ofthese devices will be given in the section pertaining to the generationof the dose gradient.

EFFECTIVE POROSITY (T) and DOSE (D) AS APPLIED TO GEL POLYMERIZATION VIAIONIZING RADIATION

Polyacrylamide electrophoresis gels are crosslinked polymer networksformed by polymerizing solutions normally containing 5-30% totalacrylamide monomer in water. Normally 2-7% of the monomer isN,N'-methylene bisacrylamide (BIS). This is added to provide cross-linksto the matrix. Normally the monomer is thermally polymerized tocompletion using standard persulfate initiator combinations, orphotochemically using riboflavin. The restrictive nature of the gel isrelated to the percent of total monomer(%T) and the amount ofcross-linker present via polymer density and degree of cross-linking.

Literature convention has defined the restrictive nature of a gel interms of the percent total monomer containing 3% BIS polymerized in thegel. For example, a gel having a "20% T porosity" demonstrates therestrictive character of a gel formed by polymerizing to completion asolution of 20% total acrylamide to which 3% BIS had been added.Therefore, one refers to gels as having 10, 15, 20% T porosity dependingon the composition.

This above convention works well in relating the electrophoreticresponse of the gel formulation since polymerization is assumed toapproach completion. In the context of the present invention, theelectrophoretic response can not be related directly to the acrylamidecontent or %T. This is because both degree and nature of thepolymerization process is controlled by the radiation exposure. Forexample, using radiation polymerization, a range of porosity can beachieved with a given initial formulation by varying the range ofelectron exposure. The equivalent porosity of an electron radiationproduced gel can be referred to as an equivalent effective %T, whichwill be termed T in the content of this disclosure.

Another term used in the disclosure, which is important to define, isthe term dose (D), which refers to that quantity of radiation energywhich is absorbed in an electrophoretic gel. The absorbed dose isdefined as the fraction of energy absorbed in a given gel of a giventhickness and the dose rate (DR) is the rate of energy absorbed in agiven gel per unit time. For example, illustrated in Example #3 is atable which identifies the fraction of incident beam radiation energyabsorbed for a given gel component of a given thickness.

EXAMPLE #3

    ______________________________________                                        THE FRACTION OF 2Mev ELECTRON RADIATION                                       ENERGY ABSORBED IN A GIVEN TARGET MATERIAL                                    OF A GIVEN THICKNESS                                                                             Mass      Fraction                                                            Thickness of Energy                                        Target Material    (t)       Absorbed                                         ______________________________________                                        Al Plate (25 mil)  .171      .382                                             Al Plate + Mylar ® (7 mil)                                                                   .178      .420                                             Al Plate + Mylar ® +                                                                         .220      .483                                             Gel (12 mil)                                                                  Al Plate + Mylar ® +                                                                         .380      .718                                             Gel (62.5 mil)                                                                Al Plate + Mylar ® +                                                                         .507      .912                                             Gel (125 mil)                                                                 Glass Plate (62.5 mil)                                                                           .349      .720                                             Glass Plate + Mylar ®                                                                        .367      .748                                             (7 mil)                                                                       Glass Plate + Mylar ®                                                                        .526      .927                                             Gel (62.5 mil)                                                                Glass Plate + Mylar ® +                                                                      .684      .995                                             Gel (125 mil)                                                                 ______________________________________                                         The source of radiation used in the above tests was the General Electric      G.E. 2Mev Resonant Transformer.                                          

The fraction of radiation energy absorbed (fa) by an electrophoretic gel12 mils thick can be calculated by subtracting the total fraction ofradiation energy absorbed for gel components consisting of; an aluminumplate (25 mils thick) and Mylar® film (7 mils thick), from gelcomponents consisting of an aluminum plate (25 mils thick), Mylar® film(7 mils) and an electrophoretic gel (12 mls thick).

From Example #3, the fraction of energy absorbed is 0.419 and 0.483respectively. Carrying out this subtraction would result in thefollowing:

    ______________________________________                                         .483                                                                         -.419                                                                          0.64                                                                         ______________________________________                                    

approximately 6% total energy absorbance is associated with theelectrophoretic gel described.

The fraction of absorbed beam energy (fa) is related to the absorbeddose rate in Mrads/sec at 1.0 milliampere total accelerator beam currentby the expression: ##EQU1## where Eo=energy of incident electron beam inMEV, I is incident beam current flux, Eo*I=power flux, fa=fraction ofpower absorbed, which is a function of sample mass thickness (td), wheretd is expressed as g/cm² =d(g/cc)*t(cm); d=density; t=sample thickness.The total absorbed dose of energy in Mrads in the gel composition isdose rate (DR(Mrads/sec)) multiplied by time (sec) This absorbed energyor dose represents the energy associated with the initiation of freeradicals in the polymerization process.

CALIBRATION OF ELECTROPHORETIC RESPONSE TO RADIATION EXPOSURE 50

Once the radiation source has been characterized, it is then necessaryto calibrate the response of the selected gel formulation for a givendose and dose rate. This calibration of electrophoretic response 50 isvery important in order to insure that the correct porosity will beproduced from a given dose and dose rate combination. The first step inthis process is to establish a mathematical relationship betweenporosity (T) and the Rf factor (Rf is defined as the protein migrationdistance divided by the distance for migration of a tracking dye whichis not restricted by the gel) for a given protein in a given gel. Thisis accomplished empirically by conducting a comparative study of themigration of well-characterized reference proteins in uniform gelscontaining various amounts of monomers polymerized to completion usingstandard persulfate initiators as well as electron irradiation.

A plot of log (Rf) versus %T is shown to give a family of linear curves,one for each different protein, as illustrated in FIG. 3. Thisrelationship is essentially a measure of the drag imposed on theproteins by the restrictive gel material. Larger proteins are slowedmore than small ones. The fitting constants relating log (Rf) to %T via:

    log (Rf)=C.sub.1 +C.sub.2 *(%T)

    %T=a.sub.1 +a.sub.2 log (Rf)

where

a₁ =-C₁ /C₂ and a₂ =1/C₂

were computed for various proteins in two types of electrophoretic gelformulations, i.e., 20T, 3C and 20T, 5C. These constants are illustratedin Example #4 below:

EXAMPLE #4

    ______________________________________                                        Fitting Constants Relating Effective                                          % T to Rf by the Function:                                                    T = a.sub.1 + a.sub.2 log(Rf)                                                 #   Protein        Mw      a.sub.1                                                                             a.sub.2                                                                              Remarks                               ______________________________________                                            Gel Type 20T, 3C:                                                         1   Insulin         3000   10.638                                                                              -27.146                                                                              BRL                                   2   Bovine Tripsine                                                                               6200   8.118 -32.288                                                                              Stan-                                     Inhibitor                           dards                                 3   Cytochrome-B(12300)                                                                          13300   6.915 -29.408                                          Sysozyme (14300)                                                          4   β-Lactoglobulin                                                                         18400   5.272 -29.790                                                                              Persul-                                                                       fate                                  5   α-Chymotrypsinogen                                                                     25700   4.676 -16.252                                                                              Poly-                                                                         merized                               6   Ovalbumin      43000   3.234 -13.284                                                                              (3% C)                                    Gel Type 20T, 5C:                                                         1   Insulin         3000   11.355                                                                              -32.925                                                                              BRL                                   2   Bovine Tripsine                                                                               6200   10.145                                                                              -28.143                                                                              Stan-                                     Inhibitor                           dards                                 3   Cytochrome-B(12300)                                                                          13300   9.226 -23.594                                          Lysozyme(14300)                                                           4   β-Lactoglobulin                                                                         18400   8.359 -19.093                                                                              Radiation                             5   α-Chymotrypsinogen                                                                     25700   7.455 -13.339                                                                              Poly-                                                                         merized                               6   Ovalbumin      43000   6.047  -9.961                                                                              (5% C)                                7   α -Lactalbumin                                                                         14400   9.550 -26.889                                                                              PHL                                   8   Tripsine Inhibitor                                                                           20100   8.278 -22.193                                                                              Stan-                                                                         dards                                 9   Carbonic Anlsydrase                                                                          30000   6.397 -18.899                                      10  Ovalbumin      43000   4.442 -16.935                                                                              Radiation                             11  Albumin (Human)                                                                              67000   3.375 -13.150                                                                              Poly-                                                                         merized                               12  Phosphorylase-B                                                                              94000   2.913 -10.124                                                                              (5% C)                                ______________________________________                                    

This study demonstrates the similarity between E-beam gels and thoseproduced by normal literature procedures. It also provides standards bywhich porosity changes produced by radiation could be related toelectrophoretic porosity values described in the literature.

Knowing the relationship of %T as a function of (Rf) for each proteinone can calculate the effective %T (T) for any gel material given an Rfvalue for a protein transported electrophoretically in that material.Therefore, the effective porosity of a gel resulting from exposure toelectrons can be measured and reported in terms of a T value using therelationship from the standards above.

Since an effective %T (T) is calculated for each protein, the effect ofradiation on the matrix as it relates to movement of individual proteinscan be checked by comparing differences between values calculated foreach protein. Also, an average effective %T value can be calculated foreach dose.

With this information established, the optimum conditions for gelpreparation can be determined. Since polymerization, termination andcross-linking rates are dependent on radical concentration which is doserate dependent, porosity (T) is expected to vary as a function of doserate (DR) at constant dose.

A general expression describing effective %T as a function of both D andDR is:

    T(D,DR)=F.sub.1 (DR)+F.sub.2 (DR)*D+F.sub.3 (DR)/D

where ##EQU2## for i=1:3.

The values of k_(in) were determined by fitting the above equations[F_(i) (DR)] to constants obtained from the data sets used to fit thefunction T versus dose for each DR for a given gel composition asillustrated in FIG. 4. These fitting constants F₁, F₂, and F₃ have beencalculated for different gels at various dose rates and are listed inExample #5 below.

EXAMPLE #5

    ______________________________________                                        FITTING CONSTANTS FOR T VS D                                                  AT VARIOUS DOSE RATES                                                                  Dose                                                                 Gel ID   Rate    RMS      F.sub.1 (DR)                                                                         F.sub.2 (DR)                                                                         F.sub.3 (DR)                          ______________________________________                                        1   AV123    0.00994 1.336  21.28  3.989  -0.752                                  AV13     0.00994 1.2071 22.65  2.303  -0.837                              2   AV4      0.01988 0.430  24.97  -1.175 -1.617                              3   AV78     0.03021 1.287  22.27  0.439  -1.545                              4   AV910    0.03976 0.887  15.95  8.460  -1.014                              5   AV1112   0.05963 1.271  9.137  18.316 -0.455                              6   AV56     0.07951 0.949  5.488  20.902 -0.141                              7   AV1314   0.09416 0.988  2.140  25.09  0.095                               8   AV1516   0.12712 0.414  1.287  23.38  0.182                               9   AV1718   0.16478 0.504  1.675  21.13  0.178                               10  AV1920   0.19538 1.227  -2.154 25.24  0.755                               11  AV21     0.22363 1.055  -7.519 25.07  1.738                               12  AV22     0.25894 0.312  7.295  2.440  -0.173                              ______________________________________                                    

This relationship allows a user to determine the resultant effectiveporosity (T) as a function of dose (D) and dose rate (DR) for a givengel. For example, referring to Example #5 if the effective porosity T isto be determined for Gel set 1 (I.D. AV123) with a dose rate cf 0.00994(Mrads/sec) the fitting constants associated with that gel and dose ratecombination are F₁ (DR)=21.28, F₂ (DR)=3.989 and F₃ (DR)=-0.752. Usingthe general expression describing effective %T as a function of dose anddose rate results in the following expression:

    T(D,DR)=21.28+3.989*D-0.752/D

Therefore, T can be calculated for any desired dose (D) by simplyinserting the value of dose into the above expression and solve for T.This relationship is used to calculate the required exposure parametersfor producing desired gradients from calibration experiments run underdifferent exposure conditions.

FIG. 5 depicts the rate of change of porosity (T) with respect to timeand dose as a function of dose and dose rate for a 20T, 5C acrylamidegels. A plot of change of T with respect to dose D, i.e. (dT/dD) as afunction of dose (D) at each dose rate (dD/dt) is shown by the upperresponse surface 104 in FIG. 5. A maximum rate of increase in T occursat a dose-rate of 0.13 Mrads/sec for gels having an initial 20T, 5Cmonomer composition. It is also seen from the upper surface 104 that therate of change of T is essentially linear with respect to dose over themaximum dose rate range. This information defines the optimum conditionsfor the most efficient use of energy for preparing gradient gels.

By multiplying dT/dD by dose rate (dD/dt), one obtains the lower surface106 in FIG. 5, which represents the rate of change of T with respect totime (dT/dt) for each dose rate. A maximum is obtained with this lowersurface 106 at a dose rate of about 0.18 Mrads/sec and corresponds tothe exposure conditions giving the maximum rate of gel production. Aswith the upper surface 104 it is seen that the response is relativelyflat at optimum exposure conditions implying a broad exposure latitude.This is important in that it reduces error in gradient production thatmay result from beam intensity fluctuations. A linear rate-dose responseis also apparent at the optimum conditions.

CALIBRATION BETWEEN RADIATION EXPOSURE AND GEL EXCLUSION LIMIT 60

In like manner to the above disclosure, a relationship between theexclusion limit molecular weight of a gel and the radiation exposureparameters can be established.

It is known from the literature and the standardization experimentsconducted that a "limiting porosity" level (pore limit) can effectivelybe reached at which proteins of a given MW no longer can migrate. SinceT (porosity) is related to dose there is expected to exist acorresponding "limiting dose" which will produce this limiting porosity.In order to produce standardized or calibrated MW gradients as functionof migration distance, it is, therefore, necessary to determine thisrelationship between "limiting dose" and exclusion limit molecularweight.

Dose exclusion limit data from sets of equivalent gels were combined andvarious mathematical functions fit to the Dose versus MW distributions.A limiting dose (LDose) versus ln(MW) relationship

    LDose=C.sub.1 +C.sub.2 * ln (MW).sub.o

gives the best fit for all sets at the optimum exposure conditionsdisclosed above. This relationship is shown in FIG. 6.

The constants C₁ and C₂ were determined for various dose rate and gelcompositions as listed below:

    ______________________________________                                                  Dose Rate                                                           Gel Types Mrads/sec     C.sub.1                                                                              C.sub.2                                        ______________________________________                                        20T 5C    .08           2.507  -0.198                                         20T 5C    .13           2.266  -0.169                                         30T 5C    .08           1.133  -0.084                                         30T 5C    .13           1.314   -0.0936                                       ______________________________________                                    

With this information the dose required to produce a desired proteinmolecular weight exclusion limit can be easily determined.

GENERATION OF DOSE GRADIENTS 80

The most common electrophoretic gel performance relationships used inthe art is molecular weight versus distance and porosity versus distanceprofiles. The accuracy by which the porosity can be controlled as afunction of distance across a gel is unique allowing for essentially aprogrammable profile which is dependent upon desired performancerelationships. With the advent of porosity control via electronradiation a gel can be manufactured to the specific needs of aparticular researcher.

The generation of the dose gradient incorporates a radiation fluxmodifying means for both fixed and conveyed or moving gels. This fluxmodifying means takes the form of moving shutters, screens, halftonefilters, grids, variable shaped apertures or electronic modulation ofthe electron flux. A typical slit used for porosity gradient productionis illustrated in FIG. 7, where depending upon the shape of the slit agiven porosity profile is achieved. For example, using a stepped doseshaped slit as a mask 108 positioned above a gel carried on a conveyormoving under a radiation source, the radiation source would polymerizethe gel 112 only in the regions of the mask which are open to the gel.If the gel were moved or conveyed relative to the mask and radiationsource, certain regions of the gel would be exposed to the radiation fora longer length of time, i.e., a larger dose. Since dose is proportionalto polymerization and cross-linking, which in turn is proportional toporosity, a pore gradient gel could be produced.

For example, in FIGS. 7 & 8, an electrophoretic gel 112 exposed toradiation using the stepped dose gradient slit 110 would have porositygradients in step fashion across the lanes 116 through 126. Lane 116would have the highest degree of polymerization corresponding to asmaller pore size 128, since the vertical slit length 130 of lane 116 isthe largest of the mask. Lane 126 would correspond to the leastpolymerized portion of the gel corresponding to a larger pore size 114.Other examples of different dose gradient masks are illustrated in FIGS.9a, b, c, d, such as a linear 130, polynomial 132, exponential 134, orstepped dose patterns 136.

Another type of flux modification device is the halftone electron filteror screen. An example of a halftone filter demonstrating a logarithmicgradient is illustrated in FIG. 10 and FIG. 11.

The halftone electron filter is essentially a plate of radiationabsorbing material 48 FIG. 11 with holes 41 located across the surfaceof the plate. The thickness of the filter material is chosen so that itwill totally absorb the electrons in the opaque regions. The holes arelocated at 1/4 inch centers, but may be closer, relative to each otherwith their diameters incrementally varying across the length (from 44 to42) of the plate. The hole diameters across the width of the plate (from42 to 43) may be equal diameter in each row. With this configuration,row 44 will allow for the passage of more electrons than row 46. Inturn, row 46 will allow the passage of more electrons than row 42. FIG.11 illustrates the electron scattering effect of the halftone electronfilter. Electrons 200 pass through the openings of the filter and arescattered 202 accordingly. If the target 206 is placed at theappropriate distance 208 from the filter 48, the diffuse transmittedbeam will produce a uniform dose pattern over the target area. Byplacing a gel underneath a halftone filter and exposing the filter to abeam of electrons the effective porosity of the gel can be made to varyacross its length according to the filter used. Therefore, a halftonefilter in conjunction with a beam of electrons provides a convenient wayof producing gradients or any other complex distributions across anelectrophoretic gel. As with the variable slit, a conveyor can be usedto produce an exposure gradient perpendicular to the travel direction.An advantage is that the beam cross section pattern can be of any shape(gaussian most likely) in the travel direction as in the case with manycommercial accelerators. With a uniform beam, a filter, screen or gridcan also be used in a fixed position relative to the sample to producethe desired 2-dimensional dose pattern.

For fixed or stationary gel exposure, porosity gradients can be createdby the use of a programmable shutter assembly which moves across the gelat a prescribed speed. The speed of the shutter as it moves across a gelfor a given dose, dose rate, and gel composition will determine theporosity profile. A detailed description of the shutter exposure systemalong with a continuous gel manufacturing process will be discussed inthe section describing production considerations 90.

PRODUCTION CONSIDERATION 90

With the system calibrated and optimized in accordance with thediscussion in the previous disclosure, it is entirely feasible toproduce gels of a given porosity profile using the batch mode orcontinuous manufacturing operation.

According to this invention the gel formulations are exposed in two partmolds having a polystyrene tray 112 and having a polyester cover sheet104 which serves as a gel support, as best seen in FIGS. 13 and 15. Atop view of FIGS. 13 and 15 are seen in FIGS. 12 and 14 respectively.The trays 112 are essentially shallow 4.5"×5" trays, 12 mils (300μ) deepand contain a series of ridges running about 3/4 of the length of thegel dividing it into eight lanes. The trays 112 are covered with 7 mil"Gel Fix"® polyester film 104 obtained from Serva Feinbiochemica toprovide a flexible support for the completed electrophoresis gel. Forspecial 2-D integrated gel application, electrodes can be printed on thesupport film. In this case the lane ridges and sample application wellsare removed from the molds.

The polyester film 104 is sealed to the polystyrene tray 112 by applyinga thin coating of Dow Corning silicone high vacuum grease to the edge ofthe mold and then rolling the two parts together. The grease provides awater-tight liquid seal. The two parts are then clamped together usingstrips of a plastic edge molding 114 for paper bundles (Slide-LockBinding Bars®) that are cut into strips the length of the gel. Followingexposure, the gel adheres preferentially to the polyester film 104support and thus can be pealed out of the mold due to the difference inadhesion between the two halves.

Uniform gel thickness is necessary to guarantee uniform electrophoreticmigration across the gel. To insure uniform thickness during exposure, a25 mil aluminum plate 102, cut to the dimensions of the gel mold, isplaced over the polyester film 104. As best seen in FIGS. 14 and 15,exposure is made directly through this plate. This is possible with oursystem because of the high energy of the electron beam. However, thealuminum plate should not be required in other manufacturing schemes.Prior to attachment to the tray 112, the polyester film 104 is squeezedonto the plate 102 using water to provide adhesion. The laminate is thenattached to the tray 112 using silicon grease and edge clamps asdesribed earlier.

The trays 112 are filled through small ports 106 located at oppositecorners of the tray 112. The trays 112 are held on an angle to allow airor argon to be displaced by the monomer solution. The ports 106 arecapped with small rubber septa 108.

The mold assembly consisting of the gel formulation 110, polyester film104, tray 112, edge molding 114, and aluminum plate 102 are placed on analuminum support 113 to provide a rigid support for the tray 112. Thisdevice is termed the gel support assembly 144 and is used for gelpolymerization regardless of the type of manufacturing process chosen.

Illustrated in FIGS. 16 and 17 is a computer driven shutter device whichis capable of producing dose gradients in a batch type mode. Theprogrammable shutter 142 is placed at that distance below the source ofionizing radiation 140, which results in a uniform radiationdistribution at the target. The gel support assemblies 144, filled withmonomer as described, are then placed in the sample holders 146 in theupright position as best seen in FIG. 16, 2 or 3 abreast. The source ofionizing radiation is then activated by opening the beam shutter 141.Simultaneously the programmable shutter 142 is activated which starts toclose via a screw driver 148 and stepper motor 150 arrangementcontrolled by the software commands of the computer 156 according to thedesired dose profile. An Intel® 8255A Programmable Peripheral interface154 is used to coordinate the analog to digital control communicationsbetween the computer and shutter drive system. To isolate the user fromhigh energy radiation, a radiation cell wall 152 made of suitableadsorption material is located between the shutter system and controlroom. A top view of this apparatus is illustrated in FIG. 17.

A second device used for gel formulation exposure is a conveyor systemfor continuous manufacturing of gels, which is illustrated in FIG. 18.The gel support assemblies 144 are filled with monomer by hand and arethen placed on a stainless steel conveyor belt 160, which is used totransport the gels to the source of ionizing radiation 164 and then onto the collection area 180. To help remove excess heat generated duringirradiation, sample cooling is provided by circulating water throughheat transfer tubes 190, which are attached to a back-up plate 192,which contacts the stainless steel belt 160 as illustrated in FIGS. 18and 19.

After placing the gel support assemblies 144 on the conveyor belt 160,the source of ionizing radiation 164 is activated, and the beam shutter166 is opened. When the beam has stabilized, the conveyor 160 is startedand the gel support assemblies 144 pass under a slit, grid or screenassembly 168 at a constant predetermined belt speed, and acceleratorbeam current.

By knowing the calibrated accelerator dependent absorbed dose rate insamples of mass thickness (td) in Mrads/ma total accelerator beamcurrent/sec from actinometry measurements, and selecting a desired doserate for the gel samples in Mrads/sec, the accelerator beam current canbe determined. With this information, knowing the relationship betweenlimiting MW and dose or porosity and dose, a specific porosity can beproduced by exposing a sample for a given length of time. Slitdimensions are normalized to a maximum dimension corresponding tomaximum desired exposure. Knowing what this maximum exposure must be andthe exposure dose rate, a belt speed can be calculated.

An illustration of a typical slit, grid, or screen assembly 168 is shownin FIGS. 20 and 21. The slit mask 196 of interest is chosen dependingupon the desired porosity profile required. The slit 196 is coupled tothe holder and shield assembly 178, which acts as a retaining means forthe electron filter of interest along with protecting the gel supportassemblies 144 from unwanted radiation. To allow the passage of theionizing radiation to the slit 196 a radiation window 198 is providedabove the slit. The slit 106 is held parallel to the shield assembly 198via four bushings 194.

The conveyor belt 160 (FIG. 18) is driven by a motor 170, which iscoupled to a reduction gear box 172, which drives a belt 174, which inturn drives a belt pulley 176. During radiation the gel supportassemblies 144 are protected from unwanted electron exposure by theshielding 178, which consists of aluminum or lead sheeting, locatedabove the conveyor 160 and sample collection area 180. As the gelsupport assemblies 144 leave the conveyor 160 they slide down an exitramp 182 into the sample storage area 180, which can be adjustedvertically by a hydraulic stand 184.

Since the electron accelerator produces a constant flux of electronswith constant energy over a defined area at a fixed rate, the dose givena specific area of the sample is directly proportional to the time ofexposure. Time can be modulated by the programmed shutter movement, orby constant conveyor movement under variable fixed slits cut todimensions corresponding to the desired gradient. Alternatively, sampleexposure can be adjusted by modulating the relative amount of incidentbeam by passing it through a grid or halftone electron filter.

The calculations of slit dimensions, shutter movement, conveyorbeltspeed, and accelerator beam current and effects resulting fromdifferences in exposure dose rate on the gel prosity are all factorswhich must be determined prior to manufacturing electrophoretic gelsusing ionizing radiation.

Modifications can be made to the conveyor system to provide continuousintroduction and removal of samples which are compatible with potentialmanufacturing processes.

An example of how each of the above manufacturing systems can be used togenerate uniform porosity exposures, separate lane porosity variationexposures, and continuous gradient exposures is described below.

Uniform Exposures

To produce a uniform exposure using the batch type shutter system (SeeFIG. 16), the samples 144 are placed on the shutter table 146 so thateach portion of the gel 144 is exposed for the same length of time. Inthe conveyor system FIG. 18, the samples 144 are passed under a squareor rectangular slit 196 at constant rate. These two manufacturingmethods are subtly different from each other even though they bothproduce the same overall dose. With the shutter system, the sample 144receives essentially a uniform instantaneous exposure over its entiresurface area simultaneously initiating polymerization at all points inthe gel. With the conveyor system a radiation front passes over the gelproducing a corresponding polymerization or reaction front, along withits associated diffusion and thermal gradients. Under the manufacturingcondition disclosed, no differences are observed in the resultant gels.

Lane Exposures

To produce a gel with separate lanes having different but uniformexposures using a batch type system (See FIGS. 16 & 17) requires themanual or automatic movement of the shutter 142 across the length of thegel in a stepped fashion to mask the sample in an exposure patternsimilar to the one described in FIGS. 7 & 8. The preferred method ofproducing "lane" exposures is the use of the conveyor system, FIG. 18,with a slit pattern the same as that described in FIG. 7.

Continuous Gradient Exposures

The final kind of exposure used was one to produce dose gradientscorresponding to a specific desired porosity range or limiting molecularweight distribution along the length of the gel in the electrophoresisdirection. These gradients were produced using both shutter and variablefixed slits with the conveyor. The use of each will be described.

Using the batch type shutter system, FIGS. 16 and 17, the shutter 142 isopened to its maximum position and the gel 144 positioned in the table146 with the electrophoresis direction perpendicular to the edge of theshutter 142. The ionizing radiation source is started, and the beamshutter 141 is opened. Simultaneously the sample shutter 142 isactivated to close at a programmed rate to give the desired doseprofile.

The preferred means of exposure is using the conveyor system (FIG. 18),with the use of masks 168 to product the gradient. The masks 168 are cutin specific patterns required to produce the dose gradients needed togenerate particular molecular weight or porosity versus distanceprofiles. Sample gels 144 are placed on the conveyor with theelectrophoresis direction perpendicular to the direction of the beltmovement and then irradiated. FIGS. 9a-d illustrates differentrepresentative mask patterns available to give desired electrophoreticporosity responses.

Illustrated in FIG. 22 is a representation of a dose profile whichyields a linear MW versus distance distribution, the results of which isillustrated in FIG. 23. Illustrated in FIG. 24 is a representation of adose profile which yields a log MW versus distance curve, the results ofwhich is illustrated in FIG. 25. In the figures representing the proteinmigration distance, the solid lines represent the theoretical predictedposition for proteins at their exclusion limit. The symbols representactual data sets of protein migratory distance.

Coating Procedure

While a casting procedure using molds must be used for making gels formpure water/monomer formulations, a continuous coating procedure can beused with solutions that have been thickened using either polyacrylamideor some other compatible, water soluble polymers. Two possible coatingtechniques will be described. The first utilizes a cover sheet, thesecond an inert coating-reaction chamber.

If the gel formulations are coated at a remote location from theradiation source, a cover sheet 210 as best seen in FIG. 28, will haveto be applied to prevent evaporation of solvent (water) and provide anoxygen barrier for polymerization. The cover sheet 210 may also containprinted electrodes and the like, or these may be included as part of thebase. To provide uniform gel thickness, spacers 212 must be used. Theseare placed on the film support 214 as a template. The monomer-polymercomposition 218 is then pumped onto the film support 214 by way of anozzle 220. A coating knife 216 is used to form the monomer-polymerformulation 218 into a uniform gel.

If the monomer-polymer composition has sufficient physical integrity, acoating of predetermined thickness can be made using a doctor knife 222,which is housed in a U-shaped bracket which provides support during gelmanufacturing as best seen in FIG. 29. As in FIG. 28, themonomer-polymer composition 218 is pumped by way of a nozzle 220 ontothe film support 214. The gel is then formed via the doctor knife 222 ndthen polymerized by the radiation source 228, in an inert atmosphere226. Temperature and humidity must be regulated to prevent solvent lossduring coating and irradiation. Both of the methods described above canbe used for irradiating thickened monomer solutions or pure watersoluble polymer solutions to produce a given gel product 224.

Monomer Conversion In Gel Materials

The toxicity of acrylamide monomer is well established. It is thereforedesirable to produce gel materials with low residual monomer content. Wehave established that the residual acrylamide content in radiationpolymerized and conventional chemical polymerized gels is essentiallyequivalent to IEF and uniform PAGE gel materials. However, radiationproduced gradient gels may contain higher residual monomer in porousregions of the gradient. Stability studies have shown, however, thatthis does not effect the electrophoretic performance or stability of thegel materials.

Analysis of the interaction of formulation and electron exposure on gelporosity suggests several approaches by which the residual monomercontent in the electron beam gradient gels can be reduced or eliminated.These include:

Use radiative cross-linking to control the restrictive properties of thegel;

Manipulation of electron dose rate to control polymer molecular weight;

Replacement of acrylamide with an acrylamide prepolymer; and

Substitution of acrylamide with non-toxic high molecular weight oligomermonomers.

Radiative cross-linking is an attractive approach for reducing theresidual monomer content in gels. This is based on our findings that (a)radiative cross-linking requires higher doses of electrons than directpolymerization and (b) that interaction of bisacrylamide with electronproduced polymer radicals leads to branching and cross-linking whichenhances the restrictive properties of the gel. By reducing thebis-acrylamide content in the gel formulation and extending the electronexposure to maximize radiative cross-linking. It is theorized that ahigher proportion of the acrylamide monomer could be consumed early inthe polymerization process without substantially increasing the porosityof the gel. The restrictive properties of the gel could then beincreased by increasing the degree of radiative cross-linking.

To demonstrate this approach, gels were prepared with reducedbisacrylamide concentrations (0.5, 1%C). The gel formulations were thengiven an expanded range (0.05 to 50 Mrads) of electron exposure. The gelporosity was then evaluated using electrophoretic separation of mixturesof standard proteins. Residual monomer content was measured by HPLC. Asshown in FIGS. 26 and 27, the gels prepared with reduced bis (1%C)display both a substantial reduction in the residual monomer content anda useful range (6-23%) of gel porosity. These findings indicate that bymanipulating the concentration of cross-linker monomer and the extent ofelectron exposure, gels can be produced with both a wide range ofporosities and a high degree of monomer conversion.

The other approaches mentioned above appear attractive as means ofreducing the monomer content throughout the entire gradient gel. We haveshown that the dose rate is an important variable influencing the extentof polymerization and thus the molecular restrictive properties of thegel. In general, gels produced at higher dose rates tend to be lessrestrictive than gels given the same total exposure at lower dose rates.At higher dose rates, monomer may be effectively consumed withoutproducing highly restrictive gels.

These observations can be used to advantage to control the residualmonomer content in pore gradient gels. Specifically by varying the doserate during the course of gel exposure, gels may be produced withessentially complete monomer consumption but with a wide range of gelporosity.

Furthermore, by replacing acrylamide with non-toxic prepolymerizedacrylamide or acrylamide oligomers, the acrylamide toxicity inelectrophoretic gels can be essentially eliminated.

Direct Production of Gels by Radiative Cross-Linking of Water SolublePolymers

Aqueous solutions of water soluble polymers may be irradiated to formrestrictive gel networks directly. This network is produced by radiationinduced cross-links on polymer molecules, as illustrated in FIGS. 26 and27. As seen in FIG. 26, representing a 20T, 1C gel composition,essentially all free monomer is consumed within the first Mrad ofexposure. A rapid increase in gel restrictiveness accompanies thedecrease in monomer corresponding to polymerization and accompanyingdirect cross-linking of the composition through the combination of BIScross-linkers and radiative cross-linking. After all monomer has beenconsumed the restrictiveness of the gel is seen to continue to increaselinearly as a function of dose. This corresponds to direct radiativecross-linking of the matrix. This response is further demonstrated bythe data plotted in FIG. 27. In each case, essentially all monomer wasconsumed with less than 1 Mrad exposure. The gels become morerestrictive at higher doses. The bottom curve corresponds to the changein porosity of linear polyacrylamide, a typical water soluble polymer.It is seen that a 20%T solution of polyacrylamide has the restrictiveproperties of a typical "6.5%T Gel." This porosity value is probably ameasure of the torturous path through which the proteins must migrate.The different curves in FIG. 27 represent compositions with differingamounts of bis-cross-linkers added to the initial formulation and showthe effect of chemical cross-linker on gel restrictiveness during theinitial polymerization stage. Radiative cross-linking generallyincreases the restrictive properties of the gel as a function of dosefor each set of formulations beyond that which can be achieved bychemical induced polymerization. It should be noted that there is asynergistic effect between radiative and chemical cross-linking duringthe early polymerization stage which results from reaction of radiationinduced polymer radicals and pendent olefin groups from incorporated BISmolecules. It is also seen from both figures that direct radiativecross-linking can extend the restrictiveness of the gel beyond themaximum attainable by normal chemical means.

As has been demonstrated here with polyacrylamide, restrictive gelnetworks also can be produced by irradiating aqueous solutions of otherwater soluble polymers. Solutions containing 10% to 30%polyvinylpyrrolidone, polyacrylic acid, polyvinylalcohol andpolymethylvinylether have been given from 1 to 50 Mrads exposures toproduce insoluble gels. The response of these systems to radiation isdependent on the chemical nature of the dissolved polymer, some beingsignificantly more sensitive than others to radiation. Other watersoluble polymers than those listed may be expected to respond similarly.

GEL PRODUCTS 100

Using this invention to induce electrophoretic gel polymerization viaionizing radiation it is possible to electrophoretically separatebioorganic molecules using the gel products 100 set forth in the abovedisclosure by the steps of placing a sample of bioorganic molecules on athin plate of the gel product and applying a voltage across a dimensionother than the thin dimension of the plate. The gel products may vary inthickness from 50μ to 2 mm with the 100-300μ range being most preferred.Also, depending upon the desired electrophoretic separation required,the gels may be designed for specific molecular weight or porosityversus distance distributions across a gel or have uniform, lane, orgradient porosity profiles. The advantages of electron beam polymerizedgels are summarized as follows:

Accurate and Reproducable Gels--The precise control of sampleformulation and preparation, electron flux and sample placement affordsexcellent reproducability in gel preparation. This translates into highreproducibility of gradient shape and type. To confirm this a series of116 gels were prepared in three different lots and exposed on threedifferent days. Gradient exposures were made using the computerprogrammed shutter (See FIG. 16). Expected error was introduced insample preparation of different lots, positioning and activation of theshutter, sample to sample variation in electrophoresis conditions and inprecise measurement of protein/tracker dye positions. The results fromthis series of experiments are shown in Example #6.

In spite of the many possible sources of error, the standard deviationof all proteins in all lots and in the combined set are essentiallyidentical and equal to 0.02. The error in protein position for the 116points is ±0.6%. The increase in CV (coefficient of variation) as theprotein molecular weight is increased from 3000 to 25,700 coupled withconstant SD indicates that the primary source of error in the experimentis in precise measurement of protein band position and not related toany chemical or structural variation between gels.

EXAMPLE 6

    ______________________________________                                        Reproducibility Statistical Summary                                           Protein                                                                               P.sub.1 P.sub.2 P.sub.3                                                                             P.sub.4                                                                             P.sub.5                                   MW→                                                                            3000    6200    13300 18400 25700 Ave.                                ______________________________________                                        Set #1                                                                        # of    28, 27  28, 27  28, 27                                                                              28, 27                                                                              28, 27                                                                              28, 27                              Points, DF                                                                    Confidence                                                                            95      95      95    95    95    95                                  Level                                                                         T value 2.052                             .sup. 2.052                         Mean    0.880   0.786   0.710 0.645 0.573 .sup. 0.719                         SD      0.021   0.021   0.019 0.020 0.021 .sup. 0.020                         CV      0.024   0.027   0.027 0.031 0.036 .sup. 0.029                         ± Range                                                                            0.008   0.008   0.007 0.007 0.008 .sup. 0.008                                                                   (0.011) =                                                                     .sup. 0.008                                                                   .sup. 0.719                         Set #2                                                                        # of    44, 43                            44, 43                              Points, DF                                                                    Confidence                                                                            95                                95                                  Level                                                                         T value 2.021                             .sup. 2.021                         Mean    0.887   0.789   0.712 0.648 0.559 .sup. 0.719                         SD      0.023   0.026   0.028 0.030 0.032 .sup. 0.28                          CV      0.026   0.033   0.039 0.047 0.058 .sup. 0.041                         ±  Range                                                                           0.007   0.007   0.008 0.009 0.009 .sup. 0.008                                                                   (0.012) =                                                                     .sup. 0.008                                                                   .sup. 0.179                         Set #3                                                                        # of    44, 43                            44, 43                              Points, DF                                                                    Confidence                                                                            95                                95                                  Level                                                                         T value 2.021                             .sup. 2.021                         Mean    0.878   0.780   0.702 0.635 0.548 .sup. 0.709                         SD      0.017   0.015   0.016 0.019 0.018 .sup. 0.017                         CV      0.019   0.019   0.022 0.030 0.033 .sup. 0.025                         ± Range                                                                            0.005   0.004   0.004 0.005 0.005 .sup. 0.005                                                                   (0.007) =                                                                     .sup. 0.005                                                                   .sup. 0.709                         Set #4                                                                        # of    116,                              116, 115                            Points, DF                                                                            115                                                                   Confidence                                                                            95                                95                                  Level                                                                         T value 1.98                              .sup. 1.98                          Mean    0.088   0.785   0.708 0.642 0.558 .sup. 0.715                         SD      0.021   0.021   0.022 0.025 0.026 .sup. 0.023                         CV      0.023   0.027   0.031 0.038 0.046 .sup. 0.033                         ± Range                                                                            0.003   0.003   0.004 0.004 0.004 .sup. 0.004                                                                   (0.006) =                                                                     .sup. 0.004                                                                   .sup. 0.715                         ______________________________________                                         Variation Between Gels = ± 0.602%    Custom Gels--Gel gradients may be     customized to any pattern porosity profile through microprocessor control     of beam, shutter, slit or mask. For example, discontinuous gradients and     gradients of complex profiles can be readily produced.

Cleaner Gels--Absence of chemical initiator obviates potential reactionwith protein samples and can reduce background staining. Separation issharper and stained protein spots show higher contrast with background.

Continuous Production of Gels--Currently, gels are batch prepared. Theinstant case allows continuous production. Each gel can thus be maderapidly under reproducable conditions.

Thinner Gels--The radiation polymerization process enables theproduction of thin gradient gels (500μ) which are not readily producedby conventional processes. Since thin gels require less power to runthey can be electrophoresed faster and are easier to handle. This isfaster and saves energy.

Reduced Endosmosis Flow--The degree of electroendosmosis flow in the gelcan be reduced due to the reduced ionic content of the gel formulation.

IEF Gels--Normal gels as prepared in the art have organic bases, such astetramethylenediamine (TMEDA) present as polymerization catalysts. ForIEF applications, such bases can migrate and distort local pH values inthe overall pH gradient. Their absence in the instant case means morestable, accurate pH gradients as well.

Safer Gels--Since the user does not have to handle toxic acrylamide, thepackaged gel materials can be more safely handled.

Faster Migration in IEF Gels--The absence of ionic initiators andhydrogen donors reduces the ionic content of the gel material. Thisallows higher voltages to be applied to the gel with reduced heatproduction.

Stable Gradient--Unreacted initiators in other systems may causepolymerization of free monomer or cross-linking of polymer to occurafter gel preparation is complete. Thus cross-link density and gradientcan change uncontrollably. Absence of initiators stabilizes thegradient.

Extended Shelf Life--Absence of thermal or photochemical, radicalinitiators potentially eliminates possible random reaction of initiatorwith free monomer, buffers, solvents, or acrylamide polymer.

What is claimed is:
 1. A porous electrophoretic gel product comprisingan aqueous-swelled porous polymer matrix formed of homo- or co-polymerswhich defines a volume, the polymer matrix being formed from watersoluble, ethylenically unsaturated monomers which can undergo radicalinitiated polymerization, the product characterized by having a constantatomic composition over its volume, an absence of a polymerizationcatalyst, being stable, having a controlled, electrophoretic resolvingcapacity that is reproducible from gel to gel for the electrophoreticseparation of charged macromolecular substances, having length, widthand thickness dimensions, and having a porosity gradient along one ofthe length and width dimensions and uniform porosity along the thicknessdimension which dimension is relatively thin.
 2. The gel product setforth in claim 1 in which the gel product is a co-polymer consistingessentially of an acrylamide and a monomer selected from the groupconsisting of N,N'-methylene-bis-acrylamide, N,N'-diallyltartramide,ethylenediacrylate, N,N'-bis-acrylylcystamine,N,N'-(1,2-dihydroxyethylene)bisacrylamide and(polyoxyethyl-trimethylolpropanetriacylate).
 3. The gel product setforth in claim 1 wherein the polymerization is initiated by ionizingradiation induced radicals.
 4. The gel product set forth in claim 1which also includes one of the group consisting essentially ofampholytes and a mixture of buffers for use in isoelectric focussing. 5.The gel product set forth in claim 4 wherein the weight/volume percentof polyacrylamide is about
 5. 6. The gel product set forth in claim 1wherein the porosity gradient is represented by a mathematical function.7. The gel product set forth in claim 7 wherein the porosity gradient inone of the length and width dimensions is discontinuous.
 8. The gelproduct set forth in claim 6 wherein the porosity gradient in one of thelength and width dimensions is logarithmic.
 9. The gel product set forthin claim 1 wherein the electrophoretic molecular resolving power isestablished by a gradient defined by said polymer matrix having varyingchain lengths, branching and cross-link densities.
 10. The gel productset forth in claim 6 which has a constant thickness lying in the rangefrom about 50 micrometers to 2 millimeters.
 11. The gel product setforth in claim 1 wherein the thickness of the gel product is constantand lies in the range from about 100 microns to about 300 microns.